The Aerobic Glycolysis Induced By HIF1?-Prohibitin Pathway In Endometriosis

Objective:Prohibitin(PHB),as well as hypoxia inducible factor-1?(HIF-1?),has been confirmed to induce the aerobic glycolysis in Endometriosis(EMs).In hypoxia,HIF-1? translocates to the nucleus,creates a dimeric complex with its partner HIF-1?subunit,and combines with the hypoxia response element(HRE)which also exists in PHB,eventually leding to the target gene transcription.In this study,we investigated the different expression of Prohibitin(Prohibitin-1,PHB),cell proliferation and apoptosis in endometrial stromal cells(ESCs)from women with endometriosis(EMs)under low oxygen tension and normal oxygen tension.To identify hypoxia-inducible factor-1?(HIF-1?)was the key transcription factor which combines with the hypoxia response element(HRE)of PHB gene to regulate the expression of PHB.Material and Methods:Endometrial stromal cells(ESCs),from women with and without endometriosis,were cultured in vitro under normoxic and hypoxic conditions.Cell proliferation and apoptosis in those cells were determined.Then we used Western blot and Immunofluorescence to quantify the expression of PHB.The core promoter area was identified by using luciferase reporter assays.siRNA transfection was used to down-regulate the expression of HIF-1?.The combination between HIF-1? and PHB was explored by the chromatin immunoprecipitation(CHIP).The transcription factor binding sites between HIF-1? and PHB gene were identified by mutational analysis.Glucose consumption and lactate generation were determined by glucose and lactate kit.Results:(1)In both 21%02 and 1%02,the proliferation rate in eutopic stomal cells(EU)was the highest compared with normal stomal cells(NE)and ectopic stomal cells(EC),and the proliferation apoptosis rate was the lowest.(2)PHB level was significantly decreased in ESCs cultured in hypoxic condition when compared with those cultured in normoxic condition.(3)Luciferase reporter assays of reporter plasmids revealed that the construct promoter activity of deletion from-1054bp to-654bp was declined significantly,and the construct promoter activity of deletion from-654bp to-254bp was increased significantly.(4)In hypoxia,the expression level of PHB protein and the promoter activity of PHB gene were significantly increased in the ESCs when down regulated HIF-1?.(5)ChIP assays showed that HIF-1? bound to PHB in hypoxia.(6)Bioinformatics confirmed that there was HRE(s)in PHB gene.Mutational analysis identified the promoter activity of PHB gene were significantly increased when HRE is mutated.(7)ESCs showed lower glucose consumption and lactate production when up regulated the level of PHB gene.Conclusion:(1)In hypoxia,the low expression level of PHB protein in ESCs may induce cell proliferation and inhibit cell apoptosis,which contrbuted to the development of EMs.(2)The positive core promoter of PHB gene was located from-1054bp to-654bp,and the negetive core promoter of PHB gene was located from-654bp to-254bp.(3)In hypoxia,HIF-1? combined with the HRE of PHB gene to down regulate the expression of PHB,which provided theoretical basis for the pathomechanism of EMs.(4)PHB gene could inhibit the aerobic glycolysis in EMs,which would provide the taget for treating EMs.(5)Further investigation needed to be done to study the role of HIF-1? and PHB in aerobic glycolysis in EMs.